Maize H3C4 promoter combined with the first intron of rice actin, chimeric gene comprising it and transformed plant

ABSTRACT

The present invention relates to a DNA sequence, a 5′ regulatory element allowing the expression of a heterologous gene in a plant cell from a monocotyledonous plant, characterized in that it comprises, in the direction of transcription, a first DNA sequence, which is a functional fragment of the sequence of the maize H3C4 promoter, and a second DNA sequence, which is a functional fragment of the sequence of the first intron of rice actin.  
     The invention also relates to a chimeric gene comprising the said DNA sequence and the plants transformed with the said gene.

[0001] The present invention relates to a new 5′ regulatory sequenceallowing the expression, in monocotyledonous plants, of a sequenceheterologous to the said regulatory sequence, encoding a protein ofinterest. The present invention also relates to a chimeric genecomprising the said regulatory sequence, a heterologous sequenceencoding a protein of interest and a 3′ regulatory sequence allowing theexpression of the protein of interest in a plant cell from amonocotyledonous plant, as well as a transformed monocotyledonous plantcomprising the said chimeric gene and the means necessary for thetransformation of plant cells and of plants.

[0002] Various promoters allowing the expression of sequences encodingproteins of interest in plants are known, are described in theliterature, and have already allowed the development, to a commercialstage, of plants modified by genetic engineering. They are promotersequences of genes which are expressed naturally in plants, inparticular promoters of bacterial, viral or plant origin such as, forexample, that of a gene for the ribulose bisphosphatecarboxylase/oxygenase small subunit (U.S. Pat. No. 4,962,028) or of agene of a plant virus such as, for example, that of cauliflower mosaic(U.S. Pat. No. 5,352,605). Promoters allowing the expression ofheterologous genes in plants are in particular described in thefollowing patents and patent applications: U.S. Pat. No. 5,086,169, EP 0353 908, 5,139,954, 5,378,619, 5,563,328, 5,589,583, 5,633,363,5,633,439, 5,633,440, 5,633,447, 5,635,618, 5,639,948 and 5,639,952.

[0003] However, some of these promoters, and more particularly thepromoters of plant origin, are not functional in monocotyledonousplants.

[0004] Arabidopsis sp. histone promoters described in patent applicationEP 0,507,698 are for example known which are particularly efficient forallowing the expression of a heterologous gene in dicotyledonous plantssuch as tobacco, oil seed rape or soya bean, which are not functional inmonocotyledonous plants such as maize.

[0005] The rice actin promoter is a promoter known to allow theexpression of heterologous genes in monocotyledonous plants (U.S. Pat.No. 5,641,876). However, the problem of identifying new functional 5′regulatory sequences for the expression of heterologous sequences inmonocotyledonous plants still remains.

[0006] The present invention relates to a new DNA sequence, a 5′regulatory element allowing the expression of a heterologous gene in aplant cell from a monocotyledonous plant, the said DNA sequencecomprising, in the direction of transcription, a first DNA sequence,which is a functional fragment of the sequence of the maize H3C4promoter, and a second DNA sequence, which is a functional fragment ofthe sequence of the first intron of rice actin.

[0007] The sequence of the maize H3C4 promoter is in particulardescribed by Brignon et al. (Plant. Mol. Biol., 22: 1007-1015, 1993). Itis the AluI fragment of the maize H3C4 promoter, of about 1 kb,corresponding to bases −7 to −1029 relative to the ATG of the sequenceencoding the maize histone H3C4.

[0008] The sequence of the first intron of rice actin is in particulardescribed in patent U.S. Pat. No. 5,641,876.

[0009] Functional fragment is understood according to the invention tomean any DNA sequence derived from the sequence of the maize H3C4promoter or from the sequence of the first intron of rice actin, whichreproduces the function of the sequence from which it is derived.

[0010] According to one embodiment of the invention, the functionalfragment of the sequence of the maize H3C4 promoter comprises the DNAsequence described by the sequence identifier No. 1 (SEQ ID NO: 1) or asequence homologous to the said sequence. Preferably, the functionalfragment of the sequence of the maize H3C4 promoter consists of the DNAsequence described by the sequence identifier No. 1.

[0011] According to one embodiment of the invention, the functionalfragment of the first intron of rice actin comprises the DNA sequencedescribed by the sequence identifier No. 2 (SEQ ID NO: 2) or a sequencehomologous to the said sequence. Preferably, the functional fragment ofthe first intron of rice actin consists of the DNA sequence described bythe sequence identifier No. 2.

[0012] The DNA sequence, a 5′ regulatory element, according to theinvention may comprise, in addition, between the first and second DNAsequences, neutral DNA fragments which are generally necessary for theconstruction of the sequence according to the invention. These are DNAfragments comprising up to 30 base pairs, preferably up to 20 basepairs. Neutral DNA fragments are understood according to the inventionto mean DNA fragments which do not substantially modify the respectivefunctions of the first and second DNA sequences of the sequenceaccording to the invention.

[0013] According to a preferred embodiment of the invention, the DNAsequence according to the invention comprises the DNA sequencerepresented by the sequence identifier No. 3 (SEQ ID NO: 3) or asequence homologous to the said sequence. More preferably, the sequenceaccording to the invention consists of the DNA sequence represented bythe sequence identifier No. 3. “Homologue” is understood according tothe invention to mean a DNA sequence representing one or more sequencemodifications relative to the reference DNA sequence described by thesequence identifier No. 1, 2 or 3, and reproducing the function of theabovementioned sequences. These modifications may be obtained accordingto the customary mutation techniques, or alternatively by choosing thesynthetic oligonucleotides which may be used in the preparation of thesaid sequence by hybridization. Advantageously, the degree of homologywill be at least 70% relative to the reference sequence, preferably atleast 80%, more preferably at least 90%.

[0014] The present invention also relates to a chimeric gene (or anexpression cassette) comprising a coding sequence as well asheterologous regulatory elements at the 5′ and 3′ positions capable offunctioning in plant cells from monocotyledonous plants, in which the 5′regulatory elements comprise the DNA sequence according to the inventiondefined above.

[0015] “Plant cell” is understood to mean according to the invention anycell derived from a monocotyledonous plant and capable of constitutingundifferentiated tissues such as calli, differentiated tissues such asembryos, monocotyledonous plant portions, monocotyledonous plants orseeds. “Monocotyledonous plant” is understood according to the inventionto mean any differentiated multicellular organism capable ofphotosynthesis, more particularly crop plants intended or otherwise asanimal feed or for human consumption, such as for example wheat, barley,oats, rice, maize, sorghum, sugar cane and the like.

[0016] According to the invention, it is also possible to use, incombination with the regulatory promoter sequence according to theinvention, other regulatory sequences, which are situated between thepromoter and the coding sequence, such as the sequences encoding transitpeptides, either single, or double, and in this case optionallyseparated by an intermediate sequence, that is to say comprising, in thedirection of transcription, a sequence encoding a transit peptide for aplant gene encoding a plastid localization enzyme, a portion of sequenceof the mature N-terminal portion of a plant gene encoding a plastidlocalization enzyme, and then a sequence encoding a second transitpeptide for a plant gene encoding a plastid localization enzymeconsisting of a portion of sequence of the mature N-terminal portion ofa plant gene encoding a plastid localization enzyme, as described inapplication EP 0,508,909. As transit peptide, there may also bementioned the signal peptide for the tobacco PR-1a gene described byCornelissen et al.

[0017] As regulatory terminator or polyadenylation sequence, there maybe used any corresponding sequence of bacterial origin, such as forexample the Agrobacterium tumefaciens nos terminator, or alternativelyof plant origin, such as for example a histone terminator as describedin application EP 0,633,317.

[0018] The coding sequence of the chimeric gene according to theinvention may comprise any sequence encoding the protein of interestwhich it is desired to express in a plant cell or a monocotyledonousplant.

[0019] This may be a gene encoding a selectable marker such as a geneconferring on the transformed monocotyledonous plant new agronomicproperties, or a gene for enhancing the agronomic quality of thetransformed monocotyledonous plant.

[0020] Among the genes encoding selectable markers, there may bementioned genes for resistance to antibiotics, genes for tolerance toherbicides (bialaphos, glyphosate or isoxazoles), genes encoding easilyidentifiable enzymes such as the enzyme GUS, genes encoding pigments orenzymes regulating the production of pigments in the transformed cells.Such selectable marker genes are in particular described in patentapplications WO 91/02071 and WO 95/06128.

[0021] Among the genes conferring new agronomic properties ontransformed monocotyledonous plants, there may be mentioned the genesconferring tolerance to certain herbicides, those conferring toleranceto certain insects, those conferring tolerance to certain diseases andthe like. Such genes are in particular described in patent applicationsWO 91/02071 and Wo 95/06128.

[0022] As regulatory terminator or polyadenylation sequence, there maybe used any corresponding sequence of bacterial origin, such as forexample the Agrobacterium tumefaciens nos terminator, or alternativelyof plant origin, such as for example a histone terminator as describedin application EP 0,633317.

[0023] The present invention is particularly appropriate for theexpression of genes conferring tolerance to certain herbicides ontransformed plant cells and on transformed monocotyledonous plants.Among the genes conferring tolerance to certain herbicides, there may bementioned the Bar gene conferring tolerance to bialaphos, the geneencoding an appropriate EPSPS conferring resistance to herbicides havingEPSPS as target, such as glyphosate and its salts (U.S. Pat. Nos.4,535,060, 4,769,061, 5,094,945, 4,940,835, 5,188,642, 4,971,908,5,145,783, 5,310,667, 5,312,910, 5,627,061, 5,633,435, FR 2,736,926),the gene encoding glyphosate oxydoreductase (U.S. Pat. No. 5,463,175),or alternatively a gene encoding an HPPD conferring tolerance toherbicides having HPPD as target, such as the isoxazoles, in particularisoxafutole (FR 95 06800, FR 95 13570), the diketonitriles (EP 496 630,EP 496 631) or the triketones, in particular sulcotrione (EP 625 505, EP625 508, U.S. Pat. No. 5,506,195). Such genes encoding an HPPDconferring tolerance to herbicides having HPPD as target are describedin patent application WO 96/38567 and in unpublished patent applicationFR 97 14264, filed on 7 Nov. 1997, whose content is incorporated hereinby reference.

[0024] Among the genes encoding an appropriate EPSPS conferringresistance to herbicides having EPSPS as target, there may be mentionedmore particularly the gene encoding a plant EPSPS, in particular frommaize, having two mutations 102 and 106, which is described in patentapplication FR 2,736,926, called hereinafter double-mutant EPSPS, oralternatively the gene encoding an EPSPS isolated from Agrobacteriumwhich is described by the sequences ID 2 and ID 3 of patent U.S. Pat.No. 5,633,435, called hereinafter CP4.

[0025] Among the genes encoding an HPPD conferring tolerance toherbicides having HPPD as target, there may be mentioned moreparticularly the HPPD from Pseudomonas and that from Arabidopsis, whichare described in patent application WO 96/38567.

[0026] In the case of the genes encoding EPSPS or HPPD, and moreparticularly for the above genes, the sequence encoding these enzymes isadvantageously preceded by a sequence encoding a transit peptide, inparticular the transit peptide called optimized transit peptidedescribed in patents U.S. Pat. No. 5,510,471 or U.S. Pat. No. 5,633,448whose content is incorporated herein by reference.

[0027] According to a preferred embodiment of the invention, thechimeric gene according to the invention comprises, in the direction oftranscription, a 5′ regulatory sequence according to the invention asdefined above, functionally linked to a sequence encoding a fusionprotein transit peptide/protein of interest, functionally linked to a 3′regulatory sequence, the different elements of the chimeric gene beingdefined above, the protein of interest being preferably an enzymeconferring tolerance to certain herbicides, more preferably enzymes ofthe EPSPS or HPPD type defined above.

[0028] Sequences encoding fusion proteins transit peptide/EPSPS, andmore particularly OTP/double-mutant EPSPS are in particular described inpatents U.S. Pat. Nos. 4,940,835, 5,633,448 and FR 2 736 926.

[0029] For the fusion protein OTP/CP4, persons skilled in the art willknow how to construct the corresponding gene by taking the sequenceencoding the CP4 described in patent U.S. Pat. No. 5,633,435 and byfollowing the procedure described in patents U.S. Pat. Nos. 4,940,835,5,633,448 and FR 2,736,926 or in the examples below. The presentinvention also relates to a chimeric gene comprising, in the directionof transcription, an appropriate 5′ regulatory sequence to ensure theexpression of a heterologous gene in a plant cell, functionally linkedto a sequence encoding a fusion protein OTP/CP4, functionally linked toa 3′ regulatory sequence. The 5′ regulatory elements comprise not onlythe 5′ regulatory elements according to the invention defined above, butalso all the appropriate regulatory elements for allowing the expressionof heterologous genes in plant cells from monocotyledonous ordicotyledonous plants which are known to a person skilled in the art orof the future, and in particular those described above.

[0030] The sequences encoding fusion proteins transit peptide/HPPD aredescribed in patent application WO 96/38567.

[0031] The present invention also relates to a cloning or expressionvector for the transformation of a plant cell or of a monocotyledonousplant, the transformed plant cells and plants containing at least onechimeric gene as defined above. The vector according to the inventioncomprises, in addition to the above chimeric gene, at least onereplication origin. This vector may consist of a plasmid, a cosmid, abacteriophage or a virus, which are transformed by introducing thechimeric gene according to the invention. Such vectors for transformingplant cells and monocotyledonous plants are well known to a personskilled in the art and are widely described in the literature.Preferably, the vector for transforming plant cells or plants accordingto the invention is a plasmid.

[0032] The subject of the invention is also a method of transformingplant cells by integrating at least one nucleic acid fragment or achimeric gene as defined above, which transformation may be obtained byany appropriate known means with the vector according to the invention.

[0033] A series of methods consists in bombarding cells or cellulartissues with particles to which DNA sequences are attached. Anotherseries of methods consists in using, as means of transfer into theplant, a chimeric gene inserted into an Agrobacterium tumefaciens Tiplasmid or an Agrobacterium rhizogenes Ri plasmid. Other methods may beused, such as microinjection or electroporation, or alternatively directprecipitation by means of PEG.

[0034] Persons skilled in the art will choose the appropriate methodaccording to the nature of the plant cell or of the plant.

[0035] The subject of the present invention is also the plant cells orplants transformed and which contain at least one chimeric geneaccording to the invention defined above.

[0036] The subject of the present invention is also the plantscontaining transformed cells, in particular the plants regenerated fromtransformed cells. The regeneration is obtained by any appropriatemethod which depends on the nature of the species.

[0037] For the methods of transforming plant cells and of regeneratingmonocotyledonous plants, there may be mentioned in particularGordon-Kamm, W.J. et al. (Transformation of Maize Cells and Regenerationof Fertile Transgenic Plants, The Plant Cell, vol. 2, 603-618, July1990), whose content is incorporated herein by reference, and thefollowing patents and patent applications: U.S. Pat. Nos. 5,177,010,5,187,073, EP 267,159, EP 604 662, EP 672 752, U.S. Pat. Nos. 4,945,050,5,036,006, 5,100,792, 5,371,014, 5,478,744, 5,484,956, 5,508,468,5,538,877, 5,554,798, 5,489,520, 5,510,318, 5,204,253, 5,405,765, EP 442174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO95/06128.

[0038] The present invention also relates to the transformed plantsderived from the culture and/or the crossing of the above regeneratedplants, as well as the seeds of transformed plants.

[0039] In the case where the chimeric gene according to the inventioncomprises a sequence encoding an enzyme conferring tolerance to aparticular herbicide, the present invention also relates to a method ofcontrolling weed in an area of a field comprising seeds or plantstransformed with the said chimeric gene according to the invention,which method consists in applying to the said area of the field a doseof the said particular herbicide which is toxic to the said weed,without, however, substantially affecting the seeds or plantstransformed with the said chimeric gene according to the inventioncomprising the said sequence encoding an enzyme conferring tolerance tothe said particular herbicide.

[0040] The present invention also relates to a method of culturing theplants transformed according to the invention with a chimeric geneaccording to the invention comprising a sequence encoding an enzymeconferring tolerance to a particular herbicide defined above, whichmethod consists in planting the seeds of the said transformed plants inan area of a field which is appropriate for the culture of the saidplants, in applying to the said area of the said field a dose of thesaid particular herbicide which is toxic to weeds should weeds bepresent, without substantially affecting the said seeds or the saidtransformed plants, and then in harvesting the cultivated plants whenthey reach the desired maturity and optionally in separating the seedsfrom the harvested plants.

[0041] In the above two methods, the application of the particularherbicide may be made according to the invention before sowing, beforeemergence and after emergence of the crop.

[0042] Advantageously, the enzyme for tolerance to a herbicide is anappropriate EPSPS, and in this case the herbicide is glyphosate or itssalts, or the enzyme is an HPPD and the herbicide is chosen from theisoxazoles, in particular isoxafutole, the diketonitriles or thetriketones, in particular sulcotrione.

[0043] The examples below make it possible to illustrate the inventionwithout seeking to limit its scope.

[0044] 1. Construction of a Chimeric Gene with a Sequence Encoding anHPPD:

[0045] The plasmids below are prepared so as to create an expressioncassette comprising a maize H3C4 histone promoter combined with theuntranslated 5′ region of the first intron of the rice actin gene (ActI)described by Mc Elroy D. et al. (Plant Molecular Biology 15: 257-268(1990)) directing the expression of the gene OTP-HPPD of Pseudomonasfluorescens.

[0046] PRPA-RD-195

[0047] The plasmid pRPA-RD-195 is a derivative of the plasmid pUC-19which contains a modified multiple cloning site. The complementaryoligonucleotides 1 and 2 below are hybridized at 65° C. for 5 minutes,followed by a slow cooling down to 30° C. over 30 minutes:

[0048] Oligo 4: 5′ AGGGCCCCCT AGGGTTTAAA CGGCCAGTCA GGCCGAATTCGAGCTCGGTA CCCGGGGATC CTCTAGAGTC GACCTGCAGG CATGC 3′

[0049] Olizo 5: 5′ CCCTGAACCA GGCTCGAGGG CGCGCCTTAA TTAAAAGCTTGCATGCCTGC AGGTCGACTC TAGAGG 3′

[0050] The hybridized oligonucleotides are made double-stranded usingthe Klenow fragment of DNA polymerase I of E. coli to extend the 3′ endsof each oligo using the standard conditions recommended by themanufacturer (New England Biolabs). The double-stranded oligo obtainedis then linked in the plasmid pUC-19 previously digested with therestriction enzymes EcoRI and HindIII and made blunt-ended using theKlenow fragment of DNA polymerase I of E. coli. A cloning vector is thusobtained which comprises a multiple cloning site so as to facilitate theintroduction of expression cassettes into a plasmid vector ofAgrobacterium tumefaciens (FIG. 1).

[0051] pRPA-RD-2010

[0052] Insertion of the sequence “H4A748 promoter-OTP-double mutantEPSPS gene” of pRPA-RD-159 into the plasmid pRPA-RD-195.

[0053] The plasmid pRPA-RD-195 is digested with the restriction enzymeSacI and dephosphorylated with calf intestinal alkaline phosphatase (NewEngland Biolabs). The plasmid pRPA-RD-173 (described in patent FR2,736,926) is digested with the restriction enzyme SacI and the DNAfragment containing the EPSPS gene is purified and linked into theplasmid pRPA-RD-195 prepared above. The clone obtained contains severalunique restriction sites flanking the double-mutant EPSPS gene.

[0054] pRPA-RD-1002

[0055] Creation of an expression cassette OTP-HPPD for use inmonocotyledonous plants. The plasmid pRP-P contains the optimizedtransit peptide (OTP) linked to the HPPD of Pseudomonas fluorescensfollowed by the polyadenylation site of nopaline synthase as describedin patent application WO 96/38567. The components of the plasmid pRP-Pare the following:

[0056] the optimized transit peptide (OTP) described in patents U.S.Pat. No. 5,510,471 and No. 5,633,448; this OTP consisting of 171 bp ofthe Helianthus annuus ribulose 1,5-bisphosphate carboxylase/oxygenasesmall subunit transit peptide (Waksman G. et al. 1987. Nucleics AcidsRes. 15: 7181) which are followed by the 66 bp of the mature portion ofthe Zea mays ribulose 1,5-bisposphate carboxylase/oxygenase smallsubunit (Lebrun et al. 1987. Nucleics Acids Res. 15: 4360) which arethemselves followed by the 150 bp of the Zea mays ribulose1,5-bisphosphate carboxylase/oxygenase small subunit transit peptide(Lebrun et al. 1987. Nucleics Acids Res. 15: 4360); the combination istherefore 387 bp;

[0057] the coding region of the HPPD of Pseudomonas fluorescensdescribed in patent application WO 96/38567; and

[0058] the nopaline synthase (nos) terminator gene (polyadenylation zoneof the nos gene isolated from pTi 37, 250 bp; Bevan M. et al. NucleicsAcids Res. 11: 369-385).

[0059] The plasmid pRP-P is digested with the restriction enzyme BstEII,treated with the Klenow fragment of DNA polymerase I of E. coli in orderto make the fragment blunt-ended, and followed by digestion with therestriction enzyme NcoI. The DNA fragment obtained, containing thecoding region OTP-HPPD about 1.5 kb, is then purified. The plasmidpRPA-RD-2010 obtained above is digested with the restriction enzymeBlpI, treated with the Klenow fragment of DNA polymerase I of E. coli inorder to obtain a blunt-ended fragment, and then digested with therestriction enzyme NcoI. The DNA fragment obtained, comprising thesequences of the plasmid vector, the H3C4 promoter combined with theuntranslated 5′ region and the first intron of the rice actin gene, ispurified and the NOS polyadenylation site is purified. The two DNAfragments purified are linked so as to create an expression cassetteOTP-HPPD comprising the maize H3C4 histone promoter (Brignon et al.)combined with the 5′ untranslated region and the first intron of therice actin gene (ActI) (Act 5′ UTR +intron 1) in order to control theexpression of the coding region OTP-HPPD incorporating the NOSpolyadenylation site (NOS polyA) (FIG. 2).

[0060] 2. Construction of a Chimeric Gene with a Sequence Encoding theDouble-Mutant EPSPS

[0061] pRPA-RD-1010

[0062] Creation of an expression cassette OTP-double mutant EPSPS foruse in monocotyledonous plants.

[0063] The plasmid pRPA-RD-109 contains the β-glucuronidase (GUS) geneof E. coli controlled by the maize H3C4 histone promoter (Brignon etal.) combined with the 5′ untranslated region and the first intron ofthe rice actin gene (ActI) described by Mc Elroy D. et al. (PlantMolecular Biology 15: 257-268, 1990). A diagram of this plasmid isrepresented in FIG. 3. The plasmid pRPA-RD-109 is digested with therestriction enzymes NcoI and EcoRI, and the large DNA fragment (about 5kb) containing the vector sequence, the GUS gene and the NOSpolyadenylation site is purified. The plasmid pRPA-RD-2010 is digestedwith the restriction enzymes NcoI and EcoRI, and the DNA fragment (about1.6 kb) containing the H3C4 promoter combined with the 5′ untranslatedregion and the first intron of the rice actin gene (ActI) is purified.The two DNA fragments purified are linked in order to create anexpression cassette OTP-double mutant EPSPS comprising the maize H3C4histone promoter (Brignon et al.) combined with the 5′ untranslatedregion and the first intron of the rice actin gene (ActI) in order tocontrol the expression of the coding region OTP-double mutant EPSPSincorporating the NOS polyadenylation site.

[0064] 3. Construction of a Chimeric Gene For Tolerance toPhosphinothricin (Bar Gene)

[0065] The phosphinothricin acetyl transferase (PAT) encoded by the bargene is an enzyme which inactivates a herbicide, phosphinothricin (PPT).PPT inhibits the synthesis of glutamine and causes a rapid accumulationof ammonia in the cells, leading to their death (Tachibana et al. 1986).

[0066] The plasmid used to introduce the tolerance to phosphinothricinas selection agent is obtained by inserting the chimeric gene pDM 302into the vector pSP72 of 2462 bp, marketed by Promega Corp.(Genbank/DDBJ database accession number X65332) and containing the genefor resistance to ampicillin.

[0067] The plasmid pDM 302 of 4700 bp has been described by Cao, J., etal. Plant Cell Report 11: 586-591 (1992).

[0068] The various components of this plasmid are:

[0069] the promoter of the rice actin gene described by Mc Elroy D. etal. Plant Molecular Biology 15: 257-268 (1990) consisting of 840 bp;

[0070] the first exon of the rice actin gene consisting of 80 bp;

[0071] the first intron of the rice actin gene consisting of 450 bp;

[0072] the region encoding the bar gene of 600 bp excised from theplasmid pIJ41404 described by White J. et al. Nuc. Acids Res. 18: 1862(1990);

[0073] the terminator of the nopaline synthase (nos) gene(polyadenylation zone of the nos gene isolated from pTi 37, 250 bp;(Bevan M. et al. Nucleics Acids Res. 11: 369-385).

[0074] 4. Transformation of Maize Cells

[0075] The particle bombardment technique is used to introduce thegenetic construct. The plasmids are purified on a Qiagen column andcoprecipitated on M10 tungsten particles according to the Klein method(Nature 327: 70-73, 1987).

[0076] A mixture of metal particles, of the plasmid pRPA-RD-1002 and ofthe plasmid of Example 3 which are described above, is then bombardedonto embryogenic maize cells according to the protocol described byGordon-Kamm, W.J. et al. (Transformation of Maize Cells and Regenerationof Fertile Transgenic Plants, The Plant Cell, vol. 2, 603-618, July1990).

[0077] 5. Regeneration and Use of the Bar Gene as Selection Agent

[0078] The bombarded calli are selected on glufosinate until greensectors appear. The glufosinate-resistant positive calli are thenconverted to somatic embryos, and then placed under conditions whichpromote germination according to the operating conditions described byGordon-Kamm, W.J. et al. (Transformation of Maize Cells and Regenerationof Fertile Transgenic Plants, The Plant Cell, vol. 2, 603-618, July1990). The young plants are transferred to a greenhouse for theproduction of seeds.

[0079] 6. Analysis of the Progeny of the Transformed Plants

[0080] The transformed plants obtained above are assumed in part to betransgenic, comprising a heterologous gene encoding OTP/HPPD conferringtolerance to isoxazoles such as isoxafutole. These transformed plantsproduced pollen, which fertilized ovules from a nontransgenic wild-typemaize. The seeds obtained are selected on sand after treating withisoxaflutole. The selection protocol is the following:

[0081] 800 ml of Fontainebleau sand are placed in a tub of sides 15×20cm. These tubs are then sprinkled with water and kept moist by supplyinga nutrient solution consisting of 5 ml of Quinoligo (Quinoline) perlitre of water. Twenty maize seeds are placed in the tubs, which arethen treated with isoxaflutole by spraying at a rate of 100 g of activematerial per hectare (300 μg of active material per tub). The tubs arethen cultured in a greenhouse. The phytotoxicity is determined 14[lacuna] after planting. According to the above conditions, thenontransformed plants exhibit 100% phytotoxicity whereas the transformedplants exhibit no phytotoxicity.

[0082] A comparative study was carried out with 20 maize linestransformed according to the invention and 20 maize lines transformedwith a corresponding gene for which the sequence encoding the firstintron of rice actin has been replaced with the sequence encoding themaize adh I intron. After treating by spraying very high doses ofisoxafutole at a rate of 200 g of active material per hectare (600 μg ofactive material per tub), the following results are obtained:

[0083] Rice actin intron according to the invention: 8/20 lines aretolerant

[0084] Maize adh I intron: 3/20 lines are tolerant The results abovedemonstrate that the combination of the maize H3C4 promoter with thefirst intron of rice actin according to the invention substantiallyenhances the expression of a protein of interest in transformedmonocotyledonous plants compared with the combination of the same maizeH3C4 promoter with another intron of the state of the art.

1 5 1021 base pairs nucleic acid single linear DNA (genomic) 1CTTATGTGCA CCATTTACTG TAATGCATAA TCATTTAATT GAATAGCAAA CTTTTCTATT 60ACTTCTTTAC TAACATAATT CTTGGTTTTA AAATTCAGTC CTCAACATTC ATTGCTCAAG 120TATAAGTTGA GACTGTCAAA ATTTACTATT TTATTTCTTC ATATTTTTTT TCCTTATACA 180CATTTTGGGC CTTACAATCC ATCATCTATA TCCATCCTTT CCGGTGTCCT CTAAAAGATT 240CCATCCTCTG AATCTTATTC CTCTCCAATA ACGTTCTCTA AATCAGGTCT CTATAAGCAA 300TACCTATATT AGAGACATTT TTTATTTTTT GTACATACAT ATTTGTCATA CTCTCAAATG 360CATTATACAT ATTTAGTTTT ACTAAACCGA TTATTTAAAG TATTCAAACG GATGAAGAAC 420TGTTTAGATA AATTCTATAT ATAGAGAATC CAGTAGCGTT CTCTAAATTT AGATGATTAT 480TTAGAGGACG CTGTTAGAAA ACGTAAAAAA TTCTTTGATT ATTTATATTT AGGGTAGAGT 540AGCCTTTATG CTTTATAGAT CTTTGGTGGA CCCAGCCTTA TACCGGTTAT TTTCGCGATT 600GCGCCTCTCA TTTTCACTCC AGCGCCCCAC ATTTTCACGT TTTCACCGAA GCGCCCAGCC 660TGCCTAACCA ACAAATTGGT ACGGTGGCGC GGTTTTCAAA AGAAGTCGGA AACCATCTGC 720ACCCACCGAC TAGTAGGCCC TCGGATCCTC CCTGATTAAG TCCTAGCCAA TAGGAGCCCA 780GAACCACCCA TCACGCGGAT CGTCCCTACG CTTCCACCTC ATCGGCGCCG TCCATCTCCA 840TCCAACACCT ATTCCGTTAC CTTGCCCATC CTCCGAAAAA ATTCTCGGCT CGCGCTCCGC 900ACCTACTACA AATACCCATC CCATCACGAC GCATCGCATC ACTGCCAAAT CCCCCAGAAA 960ATCAACACCT CCCAATTCCA CGCTGCCACC AACTCGCCGT CCTCCGCGCC AAGCACCAAA 1020 G1021 454 base pairs nucleic acid single linear DNA (genomic) 2GTAACCACCC CGCCCCTCTC CTCTTTCTTT CTCCGTTTTT TTTTTCGTCT CGGTCTCGAT 60CTTTGGCCTT GGTAGTTTGG GTGGGCGAGA GCGGCTTCGT CGCCCAGATC GGTGCGCGGG 120AGGGGCGGGA TCTCGCGGCT GGCGTCTCCG GGCGTGAGTC GGCCCGGATC CTCGCGGGGA 180ATGGGGCTCT CGGATGTAGA TCTGATCCGC CGTTGTTGGG GGAGATGATG GGGCGTTTAA 240AATTTCGCCA TGCTAAACAA GATCAGGAAG AGGGGAAAAG GGCACTATGG TTTATATTTT 300TATATATTTC TGCTGCTGCT CGTCAGGCTT AGATGTGCTA GATCTTTCTT TCTTCTTTTT 360GTGGGTAGAA TTTGAATCCC TCAGCATTGT TCATCGGTAG TTTTTCTTTT CATGATTTGT 420GACAAATGCA GCCTCGTGCG GAGCTTTTTT GTAG 454 1565 base pairs nucleic acidsingle linear DNA (genomic) 3 GAATTCCTGC AGGTCGACGG ATCCCCCTTATGTGCACCAT TTACTGTAAT GCATAATCAT 60 TTAATTGAAT AGCAAACTTT TCTATTACTTCTTTACTAAC ATAATTCTTG GTTTTAAAAT 120 TCAGTCCTCA ACATTCATTG CTCAAGTATAAGTTGAGACT GTCAAAATTT ACTATTTTAT 180 TTCTTCATAT TTTTTTTCCT TATACACATTTTGGGCCTTA CAATCCATCA TCTATATCCA 240 TCCTTTCCGG TGTCCTCTAA AAGATTCCATCCTCTGAATC TTATTCCTCT CCAATAACGT 300 TCTCTAAATC AGGTCTCTAT AAGCAATACCTATATTAGAG ACATTTTTTA TTTTTTGTAC 360 ATACATATTT GTCATACTCT CAAATGCATTATACATATTT AGTTTTACTA AACCGATTAT 420 TTAAAGTATT CAAACGGATG AAGAACTGTTTAGATAAATT CTATATATAG AGAATCCAGT 480 AGCGTTCTCT AAATTTAGAT GATTATTTAGAGGACGCTGT TAGAAAACGT AAAAAATTCT 540 TTGATTATTT ATATTTAGGG TAGAGTAGCCTTTATGCTTT ATAGATCTTT GGTGGACCCA 600 GCCTTATACC GGTTATTTTC GCGATTGCGCCTCTCATTTT CACTCCAGCG CCCCACATTT 660 TCACGTTTTC ACCGAAGCGC CCAGCCTGCCTAACCAACAA ATTGGTACGG TGGCGCGGTT 720 TTCAAAAGAA GTCGGAAACC ATCTGCACCCACCGACTAGT AGGCCCTCGG ATCCTCCCTG 780 ATTAAGTCCT AGCCAATAGG AGCCCAGAACCACCCATCAC GCGGATCGTC CCTACGCTTC 840 CACCTCATCG GCGCCGTCCA TCTCCATCCAACACCTATTC CGTTACCTTG CCCATCCTCC 900 GAAAAAATTC TCGGCTCGCG CTCCGCACCTACTACAAATA CCCATCCCAT CACGACGCAT 960 CGCATCACTG CCAAATCCCC CAGAAAATCAACACCTCCCA ATTCCACGCT GCCACCAACT 1020 CGCCGTCCTC CGCGCCAAGC ACCAAAGGAATTGGCCGCCA CCGCGGTGGA GCTCCTCCCC 1080 CCTCCCCCTC CGCCGCCGCC GGTAACCACCCCGCCCCTCT CCTCTTTCTT TCTCCGTTTT 1140 TTTTTTCGTC TCGGTCTCGA TCTTTGGCCTTGGTAGTTTG GGTGGGCGAG AGCGGCTTCG 1200 TCGCCCAGAT CGGTGCGCGG GAGGGGCGGGATCTCGCGGC TGGCGTCTCC GGGCGTGAGT 1260 CGGCCCGGAT CCTCGCGGGG AATGGGGCTCTCGGATGTAG ATCTGATCCG CCGTTGTTGG 1320 GGGAGATGAT GGGGCGTTTA AAATTTCGCCATGCTAAACA AGATCAGGAA GAGGGGAAAA 1380 GGGCACTATG GTTTATATTT TTATATATTTCTGCTGCTGC TCGTCAGGCT TAGATGTGCT 1440 AGATCTTTCT TTCTTCTTTT TGTGGGTAGAATTTGAATCC CTCAGCATTG TTCATCGGTA 1500 GTTTTTCTTT TCATGATTTG TGACAAATGCAGCCTCGTGC GGAGCTTTTT TGTAGGTAGA 1560 CCATG 1565 85 base pairs nucleicacid single linear DNA (genomic) 4 AGGGCCCCCT AGGGTTTAAA CGGCCAGTCAGGCCGAATTC GAGCTCGGTA CCCGGGGATC 60 CTCTAGAGTC GACCTGCAGG CATGC 85 66base pairs nucleic acid single linear DNA (genomic) 5 CCCTGAACCAGGCTCGAGGG CGCGCCTTAA TTAAAAGCTT GCATGCCTGC AGGTCGACTC 60 TAGAGG 66

1-21 (canceled)
 22. Fusion protein OTP/CP4. 23-35 (cancelled)
 35. Anisolated DNA sequence comprising, in the direction of transcription, afunctional fragment of the sequence of the maize H3C4 promoter and afunctional fragment of the first intron of rice actin.
 36. The DNAsequence according to claim 35 wherein said sequence of the maize H3C4promoter is the AluI fragment of the maize H3C4 promoter.
 37. The DNAsequence according to claim 35 wherein the functional fragment of thesequence of the maize H3C4 promoter comprises a sequence homologous toSEQ ID NO:
 1. 38. The DNA sequence of claim 37 wherein said functionalfragment of the first intron of rice actin comprises a sequencehomologous to SEQ ID NO:
 2. 39. The DNA sequence of claim 35 comprisinga neutral DNA fragment between said functional fragment of the sequenceof the maize H3C4 promoter and said functional fragment of the firstintron of rice actin.
 40. An isolated DNA sequence comprising a sequencehomologous to SEQ ID NO:
 3. 41. An expression cassette comprising acoding sequence and heterologous regulatory elements at the 5′ and 3′positions relative to the coding sequence capable of functioning inmonocotyledonous plant cells or monocotyledonous plants, wherein the 5′regulatory element comprises the DNA sequence of claim
 35. 42. Theexpression cassette of claim 41 wherein said coding sequence is a DNAsequence encoding a protein of interest.
 43. The expression cassette ofclaim 41, wherein the DNA sequence encoding a protein of interest is aDNA sequence encoding a selectable marker.
 44. The expression cassetteof claim 41 wherein said protein of interest is selected from the groupconsisting of a DNA sequence encoding a protein that confers herbicidetolerance, a DNA sequence encoding a protein that confers insecttolerance, and a DNA sequence encoding a protein that confers diseasetolerance.
 45. The expression cassette of claim 44 wherein said whereinthe DNA sequence encoding a protein that confers herbicide tolerance isselected from the group consisting of a DNA sequence encoding PATconferring tolerance to bialophos, a DNA sequence encoding an EPSPSconferring resistance to herbicides having EPSPS as a target, a DNAsequence encoding glyphosate oxidoreductase, and a DNA sequence encodingan HPPD conferring tolerance to herbicides having HPPD as a target. 46.The expression cassette of claim 45 wherein the DNA sequence encoding aprotein that confers herbicide tolerance is a DNA sequence encoding anEPSPS or a DNA sequence encoding an HPPD.
 47. The expression cassette ofclaim 46 wherein the DNA sequence encoding a protein that confersherbicide tolerance is a DNA sequence encoding CP4 or a double-mutantEPSPS.
 48. The expression cassette of claim 44 wherein said DNA sequenceis preceded by a sequence encoding a transit peptide.
 49. The expressioncassette according to claim 48 wherein said transit peptide is theoptimized transit peptide.
 50. An expression cassette comprising, in thedirection of transcription, a 5′ regulatory element of claim 1functionally linked to a sequence encoding a fusion protein,functionally linked to a b 3′ l regulatory sequence, wherein said fusionprotein comprises a transit peptide linked to a protein of interest. 51.The expression cassette of claim 50, wherein said protein of interest isa protein that confers herbicide tolerance selected from the groupconsisting of PAT, EPSPS, glyphosate oxidoreductase, and HPPD.
 52. Theexpression cassette of claim 51, wherein the sequence encoding a fusionprotein is a sequence encoding the fusion protein OTP/double-mutantEPSPS or a sequence encoding the fusion protein OTP/CP4.
 53. An isolatedDNA sequence encoding a fusion protein OTP/CP4.
 54. An expressioncassette comprising in the direction of transcription, a 5′ regulatorysequence functionally linked to a sequence encoding a fusion proteinOTP/CP4, optionally linked to a 3′ regulatory sequence, wherein saidexpression cassette functions in plants or plant cells.
 55. A cloning orexpression vector for the transformation of a plant cell or of a plant,which comprises an expression cassette of claim 41 and at least onereplication origin.
 56. The vector of 55, wherein said vector is aplasmid.
 57. A method of transforming plant cells, comprisingintegrating the expression vector of claim 41 into plant cells.
 58. Aplant cell which contains at least one expression cassette of claim 41.59. A transformed plant which comprises a plant cell of claim
 58. 60. Atransformed plant which is regenerated from a plant cell of claim 58.61. A transformed plant produced from the culture of a transformed plantof claim 59 or the crossing of a transformed plant of claim 59 withanother plant.
 62. A transformed seed of the transformed plant of claim59.
 63. A method of controlling weeds in a field comprising weeds andseeds or plants, said seeds or plants each comprising the expressioncassette according to claim 41, which method comprises applying to thefield a dose of herbicide which is toxic to the weeds but to which theseeds or plants are tolerant.
 64. A method of cultivating plantstransformed with the expression cassette of claim 41, which methodcomprises, sowing seeds comprising said expression cassette in a fieldcomprising weeds; cultivating plants from the seeds; applying to thefield a dose of herbicide which is toxic to the weeds but to which theseeds or plants are tolerant prior to sowing the seeds or duringcultivation of the seeds; and; harvesting the cultivated plants whenthey reach maturity.
 65. The method of claim 64 wherein said herbicideis applied before sowing the seeds, before emergence of the plants orafter emergence of the plants.